Project Summary/Abstract Hypoxia induces pulmonary vasoconstriction by increasing smooth muscle contraction and attenuating endothelium-dependent relaxation (EDR). Sustained pulmonary vasoconstriction and subsequent vascular remodeling in small distal pulmonary artery (PA) are the major causes for the increased pulmonary vascular resistance in patients with hypoxia-induced pulmonary hypertension (HPH). Endothelial dysfunction is implicated in the development of many cardiovascular diseases including HPH and diabetes. EDR is mediated via different mechanisms in small distal and large proximal arteries. Vasodilation in large proximal artery is mainly caused by endothelium-derived nitric oxide (NO) and/or prostacyclin (PGI2), while vasodilation in small distal artery is due prominently to endothelium-derived hyperpolarization (EDH). Gap junction (GJ) is an intercellular junction that transfers small molecules and propagates electric signals (e.g., hyperpolarization) to adjacent cells. GJ is more abundant in small distal artery compared to large proximal artery. We recently reported that diabetic mice exhibited significantly increased sensitivity to develop HPH and diabetic patients have higher susceptibility to develop pulmonary vascular abnormalities. It is, however, unknown whether alteration of endothelial function in diabetes is involved in determining severity of HPH. The goal of this study is to investigate a) if and how endothelial dysfunction in small distal PAs, as a result of dysfunctional GJ in endothelial cells (ECs), contributes to the development and progression of HPH and b) if and how diabetes-associated GJ dysfunction exacerbates HPH. Our preliminary data demonstrate that: i) small distal (4th-5th order) PAs, but not large proximal (1st-2nd order) PAs, exhibit great EDH-dependent relaxation that can be attenuated by hypoxia; ii) pulmonary ECs from HPH mice show lower protein level of connexin 40 (Cx40, a component of GJ) than ECs from normoxic mice; iii) hypoxia attenuates GJ activity in pulmonary ECs, iv) Cx40 overexpression enhances EDH-dependent relaxation and decreases right ventricular systolic pressure in HPH mice; v) hypoxic diabetic mice exhibit attenuated EDR in small (4th-5th) PAs compared to hypoxic control mice; and vi) pulmonary ECs from diabetic mice show lower Cx40 mRNA level than ECs from control mice. Based on these preliminary data, we hypothesize that a) chronic hypoxia downregulates Cx40 expression, attenuates GJ function, impairs EDH-dependent relaxation in small (4th-5th) PAs and, ultimately, induces HPH; and b) diabetes increases the susceptibility to HPH due to decreased GJ activity. The specific aims are 1) to identify the molecular mechanisms by which chronic hypoxia attenuates endothelial function in small distal PAs, 2) to investigate the pathogenic role of Cx40 in the development of HPH, and 3) to examine the effect of type 2 diabetes on the development of HPH with a special focus on endothelial GJ function. Completion of the proposed study will shed great lights into developing personalized therapies for diabetic patients living at high altitude and/or have hypoxic cardiopulmonary diseases.